Spore germination is essential for the major nosocomial pathogen Clostridium difficile to initiate and transmit infection, yet little is known about the molecular mechanisms regulating this complex developmental process. Since this gap in knowledge has prevented the development of therapies that can prevent dissemination of C. difficile, our long-term goal is to elucidate the molecular basis by which C. difficile spores germinate into vegetative cells. A critical step during germination is the enzymatic removal of the spore cortex, a protective layer of peptidoglycan that maintains spores in a dormant state. In the Clostridia, cortex degradation depends on the SleC cortex hydrolase being proteolytically activated by Csp family proteases. While only a single Csp protease is sufficient to induce cortex hydrolysis in Clostridium perfringens, we have shown that in C. difficile both CspC and the CspBA fusion protease are required to activate SleC upon germinant addition. Intriguingly, CspC and the CspA domain of CspBA are both pseudoproteases that we and others have shown regulate cortex hydrolysis; indeed, CspC was recently identified as a novel germinant receptor. These findings raise a number of important questions: how do pseudoproteases regulate the activity of the CspB protease? How does regulated proteolysis activate SleC? Our objective in this proposal is to determine the molecular mechanisms by which CspC, CspBA, and SleC coordinately control cortex hydrolysis. Using genetic, biochemical and structural methods, we will identify regions within the CspC and CspA pseudoproteases required for CspB activation. Targeted mutagenesis and crystallographic studies of SleC will be used to elucidate the molecular basis by which regulated proteolysis activates SleC. Lastly, interactions between CspC, CspBA, and SleC will be identified using complementary bacterial two-hybrid, immunoprecipitation, and affinity purification approaches. Collectively, the proposed studies will increase our understanding of how pseudoenzymes can control enzyme activity and how C. difficile spores sense and respond to bile salt germinants. These studies will lay the foundation for developing therapeutics that can reduce C. difficile disease transmission and recurrence.